The invention pertains to the field of fluorometers in general, and to reference systems for such fluorometers to promote stability and accuracy in particular.
In biological research it is often useful to assay samples containing trace amounts of various chemicals, hormones or enzymes that exist in the human body. In the past, such assays have been done with radioisotopes in which are called radioimmunoassays and such assays have also been done with fluorometers. Radioimmunoassays utilize radioactive isotopes to "tag" the target molecules or proteins of interest, i.e., the radioactive isotopes are attached to the molecules or proteins of interest in known tagging processes. The quantity of the molecules or proteins of interest (hereafter the targets) in the sample can then be deduced by counting the number of radioactive events which originate in the sample in a given period of time. The difficulty with this type of assay is that the radioisotopes have shelf lives which must be monitored since they lose their radioactivity over time. This reduces the effectiveness of any assay done using a weakened isotope. Further, disposing of such radioactive samples and unused radioisotopes without posing a danger to society is difficult and expensive.
Assays using fluorometers involve use of fluorescent dyes called fluorophores which fluoresce or emit light when excited with excitation light in the excitation band of the particular fluorophore being used. These fluorophores are used to tag the targets in a similar manner to use of radioisotopes to tag the target. Hereafter when the term "sample" is used, it is to be understood as referring to the sample molecules of interest as tagged with one of the fluorophores. Alternatively, sometimes the term dye will be used as a synonym for the tagged sample molecules.
Such fluorometer assays are much more desirable than radioimmunoassays since they do not use radioisotopes. However, a problem arises with these fluorometer assays where extremely small amounts of the target is present in the sample. The problem has been that the fluorometer system itself could not be made stable enough to prevent noise from degrading the answer. That is, it has been difficult or impossible to eliminate the effect on the answer calculated by the system of changes in the system other than changes in the amount of the sample concentration. Such changes, if not neutralized or eliminated, can destroy the accuracy of the answer calculated by the system.
Typically, the sample concentration of interest in fluorometer-type assays involve very low concentrations of the molecules of biological entities of interest. For example, sample concentrations of from 10.sup.-8 to 10.sup.-13 are not uncommon. To assay such low sample concentrations using fluorophores requires that a very intense light source emitting radiation having a wavelength in the excitation band of the fluorophore be used. Further, all emitted light from the fluorophore must be efficiently collected and quantized, and the effect on the system of changes in the sensitivity of the emitted light detectors over time or changing temperatures must be eliminated to get accurate results.
The desired end result is a number called the relative fluorescence intensity. This number is an indication of the sample concentration, and it is expressed in terms of a ratio. That ratio is calculated by dividing the intensity of the emitted fluorescent light by the intensity of the light from the light source used to excite the fluorophore. To do this requires that some light detector be used to detect the intensity of the emitted fluorescent light, and the same or a different light detector be used to detect the intensity of the excitation light. The two signals representing these two intensities may then be divided to arrive at the relative fluorescence intensity.
Two systems or general approaches have evolved in the prior art for generating the signals representative of the intensity of the emitted fluorescent light and the intensity of the excitation light. The first such system is illustrated in FIG. 1. This type system used a continuous light source and a single detector. Two light paths to the single detector are used. The first is the sample path which includes excitation light traveling from the light source to the sample along the excitation path through the first beam splitter and a light chopper. The emitted fluorescent light from the sample passed through the second beam splitter to the detector. The second light path is the reference path to the detector. Light on this path travels from the light source to a first beam splitter where part of it is directed downward along the reference path where it is deflected around the sample container by two mirrors and through the light chopper. This light then travels to the second beam splitter where part of it is deflected onto the face of the detector. The chopper serves to chop the continuous light beam from the light source traveling along the excitation light path and the reference light path into two light pulse trains which are interleaved in time. This interleaving is such that a pulse will illuminate the sample and cause a burst of emitted fluorescent light. This burst will be detected and a signal indicating its intensity will be generated and switched onto channel 1. Following this chain of events, a pulse of light that has traveled along the reference light path will arrive at the detector, and will be converted to an electrical signal which will be switched into channel 2. These two signals on channels 1 and 2 can be divided to derive the relative fluorescence index (hereafter sometimes referred to as the RFI).
Several difficulties exist with the system of FIG. 1. First, it is difficult to get very high intensity light in the proper wavelength out of the light source without generating a large amount of heat which shortens the life of the lamp, must be dissipated, and which may heat up the sample, thereby changing its fluorescent light emitting characteristics. Heating the sample can result in an apparent change in sample concentration even though no actual change occurred. Further, a great deal of excitation light energy is lost by being diverted down through the reference channel. Specifically, every other pulse is completely lost as the chopper blocks the excitation path and only allows light to pass along the reference path. This translates into a lower excitation efficiency in terms of the total amount of time the light is on compared to the total time the sample is being excited.
To circumvent such difficulties, the system of FIG. 2 evolved. The system of FIG. 2 used a single pulsed light source, but used two light detectors instead of one. The first light detector detected the intensity of the emitted fluorescent light from the sample. The second light detector detected the light intensity of each excitation light pulse. A beam splitter was used to divert some light energy from each excitation light pulse into the second light detector.
The system of FIG. 2 represents an improvement over that of FIG. 1. First, a pulsed light source means that higher intensity pulses can be generated by elevating the black body temperature of the light source to high enough levels to generate light of the optimal wavelength for excitation. Because this is done in a pulsed fashion, a large amount of heat is not generated, thereby minimizing sample heating, easing the heat dissipation problem, and extending the life of the light source. Since every pulse is used for excitation, the excitation efficiency is improved.
However, while the system of FIG. 2 represents an improvement over that of FIG. 1, there were new difficulties created by the system of FIG. 2. Principally, these difficulties involved the potential creation of errors in the calculated RFI caused by the changes in the system other than changes in the sample concentration. For example, it is important in the system of FIG. 2 for the characteristics of the two detectors to be matched in their drift characteristics. If the temperature of the ambient environment changes or the aging process causes the responses of the two detectors to change relative to each other in response to a single pulse, then that change will show up as an error in the RFI and an apparent change in the sample concentration when, in fact, there was no such change.
Thus, a need has arisen for a fluorometer system which solves the aforementioned problems such that stable RFIs can be computed with minimal error for sample concentrations in the range of from 10.sup.-8 to 10.sup.-13 molar concentration of the fluorophore.